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Abstract

Introduction

Skeletal muscle fiber composition and muscle energetics are not static and change
in muscle disease. This study was performed to determine whether a mitochondrial myopathy
is associated with adjustments in skeletal muscle fiber-type composition.

Methods

Ten rats were treated with zidovudine, an antiretroviral nucleoside reverse transcriptase
inhibitor that induces a myopathy by interfering with mitochondrial functions. Soleus
muscles were examined after 21 weeks of treatment. Ten untreated rats served as controls.

Conclusions

The type I to type II fiber transformation in mitochondrial myopathy implicates mitochondrial
function as a new regulator of skeletal muscle fiber type.

Introduction

Low muscle endurance and fatigue are frequent symptoms of patients with diseases that
limit the oxygen supply of muscles by its capillaries or muscular oxygen use by its
mitochondria. Muscle capillaries are lost in dermatomyositis [1], systemic sclerosis [2-4], and chronic obstructive pulmonary disease (COPD) [5], and qualitative or quantitative defects of respiratory chain components are found
in the mitochondrial myopathies [6,7]. Ultrastructural changes in mitochondria and respiratory-chain dysfunction can also
be induced by medications (statins [8], zidovudine, and other antiretroviral nucleoside analogues [9], and potentially, alcohol [10]). The physiological explanations for muscle fatigue and the adjustments of muscle
metabolism to such respiratory compromise have, however, been only poorly addressed.

In humans, most skeletal muscles are equipped with more than one fiber type to accommodate
a wide range of forces, kinetics, and endurance. Muscles specialized for maintaining
postural tone have a high proportion of fibers that contract slowly (type 1 fibers),
whereas muscles specialized for rapid movements contain a high proportion of fast-twitch
(type 2) fibers. To account for fiber-type diversity, virtually every contractile
protein of muscle fibers exists in different isoforms. Muscle-fiber types have also
developed fine-tuned systems of energy delivery, which result in diverse metabolic
profiles and oxygen requirements. Fiber types are, however, not static, as endurance
training, weight loading, or hormonal factors can promote fiber-type transformation,
even in adult muscles, by means of a coordinated antithetic regulation of fast and
slow gene programs [11-13]. No study has investigated skeletal muscle fiber-type adjustments in response to
a primary defect of the mitochondrial respiratory chain.

We therefore investigated how skeletal muscles adjust to mitochondrial dysfunction
and whether they can alter their fiber-type composition. In this study, we modeled
a mitochondrial myopathy by feeding rats with zidovudine, a nucleoside-analogue reverse
transcriptase inhibitor that impairs with the replication of mitochondrial DNA and
interferes with mitochondrial function through a variety of mechanisms, including
competition with the normal nucleotide triphosphates for incorporation into replicating
mtDNA chains, impairment of chain elongation, and excision-repair steps (extensively
reviewed elsewhere) [14]. On a global basis, zidovudine is widely used in the treatment of human immunodeficiency
virus (HIV) infections and can also cause a myopathy in humans [9,15]. Our experiments are the first to describe the ability of skeletal muscle to change
fiber-type composition by downregulating the proportion of slow fibers and upregulating
fast fibers in response to mitochondrial dysfunction. The changes in fiber-type composition
are accompanied by metabolic adjustments from oxidative to more glycolytic capacities.

Materials and methods

Animals

Male Wistar rats were purchased at Charles River (Sulzfeld, Germany), were fed a normal
rat chow (SSniff R/M-H; Spezialdiäten, Soest, Germany) ad libitum, and were housed in a normal night-day rhythm under standard conditions of temperature
and humidity. At 7 weeks of age, 10 rats received zidovudine (kindly provided by GlaxoSmithKline,
Munich, Germany) in the drinking water (100 mg/kg/d). This daily dose of zidovudine
corresponds to the human dosage adjusted for body area and the higher metabolic and
drug-disposal rate of rodents and was calculated on the basis of a daily liquid consumption
of 20 ml [16,17]. Control rats (n = 10) did not receive any zidovudine.

Observations for fluid consumption, clinical signs, and mortality were carried out
daily; body weights were recorded weekly. All rats were killed by cervical dislocation
at age 28 weeks, immediately before organ collection and postmortem examination. Soleus
muscle was snap frozen and cryopreserved in liquid nitrogen until subsequent analysis.
Muscle aliquots were fixed in glutaraldehyde (3%) for subsequent electron microscopy.
Serum was collected by puncture of the Venae saphenae laterales [18] before cervical dislocation in anesthesia with isoflurane (Abbott, Wiesbaden, Germany).
All animal work was performed after animal welfare board approval (Regierungspräsidium
Freiburg; Department 3, Nr. 35/9185/.81/G-07/67) and conformed to institutional guidelines
as well as to the NIH policy [19].

Histopathology and mitochondrial ultrastructure

Soleus muscle-fiber diameters were morphometrically quantified in all rats on three
randomly selected 0.09-mm2 areas of 8 μm thick, hematoxylin and eosin-stained sections, by using an automated
image-analysis and processing software (Leica QWin Standard v2.7; Leica Microsystems,
Imaging Solutions, Cambridge, UK). The histochemical assay for myofibrillar ATPase
activity (pH 4.35 or 10.5) was used to distinguish and morphometrically count fast
and slow muscle fibers [20]. On 4-μm cryostat muscle transverse sections, succinate dehydrogenase (SDH) and cytochrome
c-oxidase (COX) histochemistry was performed [21]. The evaluating person was blinded to the group status of all animals. Two randomly
selected soleus muscle samples from each group were examined with electron microscopy,
as described [22].

Respiratory-chain enzyme activities

Histochemical COX and SDH staining is difficult to quantify reliably. We therefore
measured the activities of COX, SDH, and nicotinamide adenine dinucleotide hydrogen
dehydrogenase (NADH-DH) in freshly prepared soleus muscle extracts with spectrophotometric
assays, as described [23]. NADH-DH and COX are the multisubunit complexes I and IV of the mitochondrial respiratory
chain and are encoded partly by nuclear DNA (nDNA) and partly by mtDNA, whereas SDH
is a respiratory chain component (complex II), which is encoded entirely by nDNA.

Single-fiber mtDNA copy numbers

In each animal, three fast and three slow fibers were picked with a microcapillary
under an inverted microscope from a 14-μm-thick, ATPase activity (pH 10.5) typed,
transverse soleus muscle section [24]. Total DNA from single fibers was released with 5 μl of a solution containing 200
mM KOH and 50 mM dithiothreitol (incubated for 1 hour at 65°C), followed by a neutralizing buffer (5
μl) containing 900 mM Tris-HCl, pH 8.3, and 200 mM HCl [24]. MtDNA and nDNA copy numbers were quantified from 2 μl of the solute by quantitative
PCR, as described [25]. Amplifications of mitochondrial and nuclear products were performed in triplicate.
Absolute mtDNA and nDNA copy numbers were calculated by using serial dilutions of
plasmids with known copy numbers.

Microarray analysis

RNA was extracted from eight randomly selected frozen muscles from each group with
the Uneasy Kit (Qiagen, Hilden, Germany). Quantity and integrity of the RNA were verified
by using RNA 6000 nano chips (2100 Bioanalyzer; Agilent, Palo Alto, CA, USA). RNA
samples (500 ng) with an RNA integrity number of greater than 9 were further processed
with the GeneChip Whole Transcript Sense Target Labelling Assay from Affymetrix (Santa
Clara, CA, USA) according to the manufacturer's instructions.

Arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G, and raw data were
imported into the Refiner module of Genedata Expressionist software (Martinsried,
Germany, version 5.3.5), in which quantile normalization and probe summarization was
performed by using its Refiner condensing algorithm [26]. The microarray data were uploaded (ArrayExpress accession number: E-MEXP-3642) in
the ArrayExpress Archive [27].

Statistics

The Kolmogorov-Smirnov test was used to analyze for normal distribution. Groups were
then compared with ANOVA, Mann-Whitney, unpaired t test, or Wilcoxon analysis, as appropriate. Skewed data are provided as median plus
interquartile ranges (IQRs), and normally distributed data, as group means and standard
deviation (SD). Correlations were computed as nonlinear exponential regressions. All
graphics and calculations were performed by using the Sigma Plot 2000, version 8.0
(SPSS, Inc.) and the Sigma Stat, version 3.1 (Jandel Inc.) packages.

To identify differentially expressed genes between the groups in microarray analysis,
the unpaired Bayes T test (CyberT) [26] with the Bayes confidence estimate value set to 24 and a window size of 101 genes,
as well as 100% valid values in each group, was performed with the Analyst module
of Expressionist. To estimate the false-discovery rate, the Benjamini-Hochberg q value was calculated in a sequential Bonferroni-type procedure [28]. We then used the "N-fold regulation" activity of Analyst to calculate the median
ratio between the experimental groups. Only genes from the categories "main" and "unmapped"
(see Affymetrix transcript annotation RaGene-1_0-st-v1.na30.1.rn4.transcript) were
included, thereby omitting control probes or genes with uncertain annotation. The
false-discovery rate, which estimates the number of false positives within a list
of significant genes, was chosen as 10%.

Results

Zidovudine induces a respiratory-chain myopathy

The daily fluid consumption and body weight of the rats was unaffected by zidovudine
(data not shown). The autopsy did not reveal macroscopic organ anomalies. Soleus muscle
fiber diameters were decreased in the zidovudine group (Figure 1, Table 1). After 28 weeks, groups did not differ in serum levels of creatinine kinase, resting
lactate, and glucose. Serum creatinine levels, however, were lower in rats treated
with zidovudine, indicating reduced muscle mass (P = 0.001) compared with untreated rats. Electron microscopy revealed a focal disarray
of the myofibrillar lattice in the zidovudine group (Figure 2). The crystal architecture was lost in a substantial proportion of the organelles
and contained deposits of electron-dense material. Mean mtDNA copy numbers were decreased
by 29% (P < 0.001) in zidovudine-treated rats compared with control animals (Table 1). Histochemical COX/SDH staining showed a uniformly downregulated respiratory-chain
activity and no clear fiber type-specific pattern. NADH-DH and COX activities in the
soleus muscle were depressed in the zidovudine group (P = 0.042 and P = 0.026, respectively; Table 1). In contrast, the activity of SDH was unaffected (P = 0.7; Table 1). These data indicate that zidovudine induced a metabolic myopathy with depleted
mtDNA copies and a specific downregulation of mtDNA-encoded respiratory chain activities
and consecutive fiber atrophy.

Investigating metabolic adjustments, we found nucleus- and mtDNA-encoded respiratory
chain subunits to be coordinately downregulated in zidovudine myopathy, although many
changes were not statistically significant. The transcription of the rate-limiting
enzymes of glycolysis and glycogenolysis was enhanced (Table 2) and the mitochondrial carnitine shuttle (carnitine palmitoyltransferase, CPT1b), and β-oxidation (3-hydroxy-acyl-CoA dehydrogenase) downregulated.

Thus, mitochondrial dysfunction is associated with a coordinate regulation of a multitude
of transcription factors that orchestrate the transformation from type I to type II
fibers.

Discussion

The present study demonstrates a previously undescribed skeletal muscle fiber-type
transformation from slow fibers to fast fibers in a mitochondrial myopathy. The changes
in fiber-type composition occur in the absence of muscle regeneration and not only
are demonstrated at the level of myosin heavy-chain isoforms and isoforms of other
contractile proteins, but also are paralleled by adjustments in the metabolic profile
and a switch from an oxidative to a more-glycolytic transcriptosome. From a mechanistic
perspective, this response of muscle energetics to the primary defect in respiratory
chain function may maintain muscle strength via increased recruitment of glycolysis
for ATP production, at the expense of increased energetic cost. The switch to more-glycolytic
type II fibers, which are characterized by an increased lactate production compared
with type I fibers, could contribute to the hyperlactatemia observed in patients with
mitochondriopathies [36]. The fact that hyperlactatemia is typically observed only, or at least is aggravated
during exercise in patients with inherited mutations in mtDNA [37,38] can explain the normal lactate levels in our rats in whom blood was collected at
rest. Fiber-type switching is also observed in conditions associated with impaired
blood oxygenation [39] and diminished muscle microcirculation [40].

In COPD, muscle hypoxia is associated with an increased proportion of type II fibers
[39,41], a reduced number of mitochondria [42], increases in glycolytic enzyme activity, and an impairment of oxidative capacity
[43].

Patients with idiopathic inflammatory myopathies also reveal an increased proportion
of fast fibers and a lower proportion of slow fibers compared with healthy controls
[40]. Even in healthy humans exposed to high altitude, the proportion of type I fibers
is decreased [44,45]. Although we failed to identify a single master switch of type I to type II fiber
transformation, these observations indicate that an impairment of mitochondrial respiration
of many causes promotes type II fiber formation.

Interestingly, slow fibers showed even less mtDNA content than did fast fibers in
zidovudine-treated rats. This observation may be explained by the dynamics of the
system (for example, the possibility that these slow fibers could still be in the
process of converting). Alternatively, this finding could be explained with a physiologically
higher mtDNA turnover in slow (oxidative) fibers compared with fast (glycolytic) fibers,
and therefore an increased susceptibility to the inhibition of mtDNA replication conferred
by zidovudine. Clearly, regulators of fiber type exist in addition to mtDNA content,
and vice versa. Downregulation of the ERRγ may explain some of the biologic processes
observed in our model, as ERRγ physiologically promotes a switch to slow muscle fibers
and induces oxidative metabolism by increasing mitochondrial number, size, and functions
[35].

Type II fibers in our study had a higher degree of atrophy than did type I fibers,
despite the fact that the former appeared to be less dependent on mtDNA replication
than the latter, as evidenced by a lesser degree of mtDNA depletion. Because muscle
disuse affects mainly type I fiber diameters [46,47], the type II fiber atrophy in our model suggests a mechanism related to mitochondrial
dysfunction. This hypothesis is further supported by the predominant type II fiber
atrophy in other conditions associated with muscle hypoxia, such as COPD [41], systemic sclerosis [2-4,48], and inflammatory myopathies [5]. Age-related sarcopenia is also associated with a predominant atrophy of type II
fibers and an increased abundance of fast myosin heavy-chain isoforms in soleus muscle
[49]. It is interesting to speculate, whether mitochondrial dysfunction, which has also
been implicated in aging, may be a driver of these characteristics of the aging muscle
[50]. Myostatin has been described as a potent negative regulator of muscle mass, and
increased myostatin expression is particularly associated with type II atrophy [51]. Consistent with this, we found a fourfold enhancement of myostatin transcription
(Table 2). Sarcopenia, in combination with the disabled aerobic energy supply of slow-twitch
fibers, can also explain muscle weakness on static and dynamic exercise, fatigue and
muscle atrophy observed in patients with mitochondrial myopathies [52].

The effects of mitochondrial dysfunction and hypoxia on fiber-type composition have
important clinical implications for training and rehabilitations programs by suggesting
that exercise intolerance in mitochondrial dysfunction may be improved not only by
cardiopulmonary mechanisms, but also by promoting fiber type II formation, either
by resistance training, or pharmacologically by targeting the calcineurin-dependent
nuclear factor of activated T-cells (NFAT) with calcineurin inhibitors [53,54].

In mitochondrial myopathies, muscle strength and oxidative capacity were improved
without type I fiber enhancement [55,56], and in COPD, muscle strength and oxidative capacity were enhanced without alterations
in lung function [41,57]. In idiopathic inflammatory myopathy, however, endurance training increased type
I fiber proportions and diameters [40]. This difference could be explained by the preservation of mitochondrial function
in idiopathic inflammatory myopathy, which enables type I fiber formation, and the
impairment of mitochondrial function in inherited or acquired defects of the mitochondrial
genome, which disables type I fiber formation. Clearly, more research is needed about
the different effects of training programs on cardiopulmonary function, skeletal muscle
microcirculation, oxidative capacity, and fiber-type composition in these different
conditions.

Conclusions

Our work demonstrates a type I to type II fiber transformation in a mitochondrial
myopathy and a preferential atrophy in type II fibers. The skeletal muscle fiber-type
transformation in the absence of fiber-type regeneration and observed adjustments
from oxidative to glycolytic metabolism provide evidence for mitochondrial function
as a new regulator of skeletal muscle fiber type and other metabolic capacities. The
effects of mitochondrial dysfunction on fiber-type composition have important clinical
implications for training and rehabilitations programs.

Competing interests

The authors have no competing interests.

Authors' contributions

DL and ACV carried out the experiments, participated in the design of the study and
participated in writing the manuscript. DP performed the microarray analyses. NV and
UAW conceived the study, participated in its design and coordination, analyzed the
data, and wrote the manuscript. JBK participated in muscle-fiber analyses. EB carried
out the blood analyses. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by DFG grant VE 492/2-1. The article-processing charge was
funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg
in the funding program Open Access Publishing. We also thank Karin Sutter and Carmen
Kopp for expert technical assistance.